Artificial chimeras engineered to simulate multiple biological threat agents

ABSTRACT

This invention provides safe, non-infectious chimeras that include the nucleic acid signature of most bacterial and viral biological threat agents. These chimeras mimic properties of threat agents and are useful as simulants to develop, evaluate, test, and train on nucleic acid-based biodetectors and diagnostic products of interest in biodefense, without the need for accessing or producing virulent agents.

RELATED APPLICATION

This application is a divisional of application Ser. No. 12/177,527,filed on Jul. 22, 2008, now U.S. Pat. No. 7,790,452.

FIELD OF THE INVENTION

This present invention includes the design and construction ofnon-infectious chimeras that include the nucleic acid signature of mostbacterial and viral biological threat agents. One of the engineeredchimeras simulates the biological threat agents whose genomes are DNAand the second engineered chimera simulates biological threat agentswhose genomes are RNA. The chimeras of the present invention are alsoincluded in methods and devices of the present invention such as nucleicacid-based biodetectors and diagnostic products, and as simulants toallow the safe validation (and to compare) the performance oftechnologies, products, and devices used in biodefense, as well as inclinical detection and diagnosis of the said agents

BACKGROUND OF THE INVENTION

The threat of biological warfare has existed for centuries. Bydefinition, biological warfare involves any deliberate use of disease toattack humans, plants, animals, or infrastructure. Biological weaponshave been used only occasionally, but they have the potential to inflictgreat harm. Unlike the materials necessary to produce nuclear weapons,microorganisms, toxins, and viruses that are dangerous to human, animal,and plant life can be found abundantly in nature. The technology neededto turn these agents into weapons is less sophisticated than what isnecessary to develop nuclear weapons. Furthermore, only a very smallquantity of material is needed, much less than that needed to producenuclear weapons, but could potentially cause a comparable death-toll.

Technology allows for some biological threat agents, which in theirnatural state pose only minimal dangers, to be genetically engineeredinto more threatening forms. Their availability in nature also changes,and science continues to discover new biological threat agents. TheCenter for Disease Control (CDC) and other agencies have compiled a listof the biological agents of greatest concern. They are segregated intocategories, depending on a variety of factors.

Though the need to develop biological defense technologies to protectagainst the threat of terrorism is increasing, such biological defensetechnologies are hard to develop and test. Biological defensetechnologies are successful if they are able to detect the biologicalthreat agent, inhibit biological threat agent contact with its host,inhibit biological threat agent growth, or kill the biological threatagent. Developing and testing biological defense technology in thepresence of a biological threat agent poses serious hazards. Exposure ofpeople working on defense technology, and/or the population at large, toa biological threat agent may result in serious injury or death. Methodsallowing the safe development, testing, and training of biologicaldefense technology are needed to minimize, or eliminate, the potentialhazards associated with such technology development. However, the use ofactual virulent threat agents is costly and risky. Furthermore,development and testing of technologies dealing with more than onethreat agent face almost insurmountable difficulties in producing,storing, and employing more than one threat agent simultaneously.

The use of biological threat agents in the development, testing, andtraining of biological defense technology is impaired by safety issues,high cost, the need of special infrastructure and uncommon expertise. Asimulant is an agent having biological and/or physical characteristicssimilar to a biological threat agent but when used in place of thebiological threat agent is not harmful. Though the use of methodsinvolving simulants is a good idea, very few simulants have beenidentified and are being used. In biodefense a few simulants, includingspores of Bacillus subtilis (as surrogate of B. anthracis), Pantoeaagglomerans (as surrogate of all vegetative threat bacteria) and thephage M13 (as surrogate of all threat viruses), are used in methodsdevelopment, training, and testing and evaluation of biodefensecountermeasures, and equipment. These simulants are totally inadequateto simulate threat agents on nucleic-acid based technologies, since B.subtilis, P. agglomerans, and M13 do not share genes with any of theactual threat agents that they are intended to mimic

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to meet theforegoing needs by providing safe methods for the development, testingand training of biological defense technology. One embodiment of thepresent invention is a chimera comprising a plurality of segments,wherein each segment uniquely corresponds to a portion of the genome ofa threatening biological agent wherein the genome is DNA. It ispreferred that the threatening biological agent is selected from thegroup consisting of: Bacillus anthracis, Yersinia species, Burkholderiaspecies, Francisella species, Brucella species, Coxiella bumetii,Ricketsia species, enterohemorrhagic Escherichia species, and variolavirus and the chimera further comprising a nucleic acid sequencecomprising SEQ ID NO. 12. It is also preferred that the chimera of thepresent invention includes a segment having a DNA sequence derived fromSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

Another embodiment of the present invention includes a chimeracomprising a plurality of segments, wherein each segment uniquelycorresponds to a portion of a genome of a threatening biological agentwhose genome is RNA. It is preferred that the threatening biologicalagent is selected from the group consisting of: Eastern EquineEncephalitis Virus, Junin virus, Marburg virus, Dengue virus, VenezuelanEquine Encephalitis Virus, Crimean Congo virus, Influenza virus, RiftValley Fever Virus, Machupo virus, Lassa virus, and Yellow Fever virus,and the chimera further comprising a nucleic acid sequence comprisingSEQ ID NO.26. It is also preferred that this chimera of the presentinvention includes segments of DNA sequences derived from SEQ ID NOs:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.

Another embodiment of the present invention includes a vector comprisinga chimera of the present invention. The vector may be a plasmid, avirus, a cosmid, or a yeast artificial chromosome. Preferably the vectoris a plasmid or a virus.

Another embodiment of the present invention includes a method of testinga detection technology, comprising the steps of: (a) providing a samplecontaining the chimera of the present invention in lieu of a samplecontaining a biological threat agent; and (b) using said detectiontechnology in accordance with normal or standard procedures to detectthreat agent in the sample; and (c) determining the effectiveness ofsaid detection technology in detecting a portion of the chimera. It ispreferred that the detection technology comprises a nucleic acid probecapable of selectively hybridizing to at least a portion of a chimera ofthe present invention. It is also preferred that this method of thepresent invention also comprises the step of measuring a level ofdetectable signal.

In yet another embodiment of the present invention, the chimeras of thepresent invention may be used as positive controls when conductingassays for detection of biological threat agents in samples. Forexample, if ten different samples suspected of containing threat agentwere being tested to detect a biological threat agent, an eleventhsample containing a chimera of the present invention could be testedconcurrently to ensure that a positive test result is obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the presentinvention and, together with the description, explain the advantages,and principles, of the invention.

FIG. 1 Selection of Nucleic Acid Segments using Bioinformatics

FIG. 2 Design and Synthesis of Nucleic Acid Segments for DetectingBiological Threat Agents having DNA Genomes

FIG. 3 Design and Synthesis of Nucleic Acid Segments for DetectingBiological Threat Agents having RNA Genomes

FIG. 4 A Plasmid Containing the Chimera for Detecting Biological ThreatAgents Having DNA Genomes.

FIG. 5 A Plasmid Containing the Chimera for Detecting Biological ThreatAgents Having RNA Genomes

FIG. 6 Confirmation of simulant construct by release of biothreat-agentspecific bands by restriction enzyme digestion and gel-electrophoresisanalysis

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made to preferred embodiments of this invention.The current invention relates to biological threat agent simulants andto methods and products in which simulants replace biological threatagents during the development, testing, and/or training of biologicaldefense technology. In order to better understand the invention, thefollowing terms have been defined.

The term “biological defense technology” means a device, product and/ormethod able to detect a threatening biological agent, protect people,plants, livestock or other assets from contact with a threateningbiological agent, and/or render harmless one or more threateningbiological agents. Examples of biological defense technology includefilters, masks, protective clothing, protective creams or gels,decontamination products and solutions, and devices or methods to detectand/or identify threat agents. A device includes a machine and/orequipment. A product includes a filter, gel, foam or othernon-mechanical item. A method includes the use of a product and/ordevice.

The term “harmful” means resulting in injury, disease or death.

The term “inactivate” means to kill threat agent organisms, cells,spores or viruses and render them harmless or nonviable.

The term “virion” means a budded virus, or a virus not enmeshed in apolyhedrin matrix.

The term “simulant” means an agent having similar biologicalcharacteristics to a threatening biological agent but when used in placeof the threatening biological agent is not harmful. The term includesone or more simulants and/or any combination of simulants.

The term “threatening biological agent” or “biological threat agent”means microorganisms, toxins, and/or viruses that are dangerous tohuman, animal, and/or plant life and as defined in this patentapplication. The term includes one or more threatening biological agentsand/or any combination of threatening biological agents.

The term “virus threat agent” means a threatening biological agent thatis a virus dangerous to human, animal, and/or plant life.

A simulant of the present invention is one or more agent(s), such as anucleic acid sequence, preferably a DNA sequence that corresponds to oneor more threatening biological agents. Such a simulant of the presentinvention takes the place of one or more threatening biological agentsduring the development, testing, and training of biological defensetechnologies.

Specifically, the simulant(s) of the present invention are chimeras; agenetic element made up of a plurality of nucleic acid segments, whereineach segment corresponds to the nucleic acid sequences of a threateningbiological agent. The chimerical simulants are by design non-infectiousto humans. Threatening biological agents are described within the Centerfor Disease Control (CDC) list of today's most dangerous biologicalagents, that is, within Category A, Category B, and/or Category C of thelist. The CDC's list of the most dangerous biological agents includesorganisms such as anthrax, plague, smallpox, tularemia, and viralhemorrhagic fevers.

The present invention specifically includes design and construction bygenetic engineering of non-infectious chimeras that include the nucleicacid signature of most or all bacterial and viral biological threatagents. One embodiment of the present invention is chimeras thatsimulate biological threat agents whose genomes are DNA. Examples ofbiological threat agents whose genomes are DNA include: Bacillusanthracis (signatures from both virulent plasmids), Yersinia species,Burkholderia species, Francisella species, Brucella species, Coxiellaburnetii, Ricketsia species, enterohemorrhagic Escherichia species, andvariola virus (Smallpox). Another embodiment of the present invention ischimeras that simulate biological threat agents whose genome is RNA.Biological threat agents whose genome is RNA include members of thearenaviruses, filoviruses, alphaviruses, flaviviruses, and hantaviruses,more particularly the viruses: Ebola, Lassa, Yellow fever, EasternEquine Encephalitis, Junin, Marburg, Dengue, Crimean-Congo, VenezueleanEquine Encephalitis, Rift Valley Fever, Machupo, and Influenza. TheChimeras once identified have been cloned into vectors such as viruses,plasmids or any other vehicle that allows the storage and amplificationof the chimera sequences.

The risk of human injury or death is minimized when a simulant is usedin the place of a threatening biological agent during the development,testing and/or training of a biological defense technology. Because thesimulant and the threatening biological agent are selected to havesimilar characteristics (corresponding nucleic acid sequences) with thesimulant being non-pathogenic, a simulant of the present invention maytake the place of a threatening biological agent for productdevelopment, testing and evaluation, training, as positive controls, andwherever a non-infectious surrogate can beneficially replace actualthreat agents. The results generated from such development, testingand/or training of a biological defense technology are then used tocreate new and effective biological defense technology, or improveexisting biological defense technologies.

Discussion will now focus on examples of biological defense technologyand their functions. Biological defense technology able to detect athreatening biological agent includes devices, products, and/or methodsable to detect such agents in the air, in water, in food, in bodilyfluids, or on solid surfaces. Detection of such agents in air generallyconsists of three steps: sample collection; sample processing; andsample analysis. Instrumentation accomplishing each step may be part ofan integrated system, or samples may be collected, processed, andanalyzed by separate systems (or by humans working with laboratoryequipment). Some detection systems may sample the air passively, usingcurrents in ambient air to cause airborne agents to move into theportion of the device that performs the analysis (in much the same wayas a smoke detector detects smoke particles only when particle-laden airwafts into the interior of the detector).

Most active samplers that draw agents from air exploit one or morephysical characteristics of the agents targeted for collection andcontact with the biological defense technology. Such methods include butare not limited to the use of filters causing separation of particlesfrom air based on size. Air can be drawn by fans (or other methods ofmoving air) and passed through filters designed with pore sizes smallenough to retard the passage of airborne particles that carry virions.Another class of samplers accelerates air (and therefore airborneagents) and increases the momentum of airborne agents, then passes suchparticles through a path in the instrument in such a way that themomentum of particles causes them to leave the airstream and impact on asurface or into a fluid where they are arrested. Such devices are oftensaid to work by “impaction” and may be called an “impaction sampler”.Conceivably, air samplers for threatening biological agents could alsowork by adsorption (an adsorption sampler), in which air is passedthrough a column filled with a porous substrate that has an affinity forthe threatening biological agents based on one or more methods,including but not limited to: charge, the complementarily of molecularsurface structures (including but not limited to an antibody-antigeninteraction), relative hydrophobicity/hydrophilicity. Sample collectionfrom liquid samples employs many of the same techniques listed above.

Sample collection from surfaces usually employs the use of a swab (oftencomposed of cotton, but can be any of a large number of materials) orother material or device that is wiped over a surface with the intentthat particles on the surface adhere to the swab. Samples from food caninvolve the use of swabs or a more frequently a disruption of a portionof the food into a proper media and further analysis. Collection ofsamples from bodily fluids, including sputum, bronchial swabs or lavage,urine, feces, spinal fluid, or blood, is well known to those involved inthe art.

The term “sample processing” refers to methods of preparing a sample foranalysis, which is making the threatening biological agent or componentsthereof such as membrane proteins, DNA, and/or RNA accessible (able tocome in contact with) to a detection device so that the detection deviceis able to detect the presence of a molecule characteristic to abiological threatening agent. Such molecules include RNA, DNA, proteinand/or lipid (i.e., content and/or composition). Typically, theintegrity of a threatening biological agent's cell, spore, or virion isdisrupted by chemical, enzymatic, electrical, mechanical and/or othermeans. For example, such disruption means may cause the release ofnucleic acids from a threatening biological agent and make themavailable for methods of analysis that rely upon nucleic acid sequenceinformation for detection and identification. Another reason a samplemight require preparation is that a molecule characteristic of athreatening biological agent may have to be modified or combined withother compounds before analysis. An example of this kind of modificationis the derivatization of small molecules before gas chromatographicanalysis.

A biological defense technology may detect a nucleic acid signature of athreatening biological agent. Nucleic acid hybridization is used todetect a biological agent by contacting a target nucleic acid (i.e. thenucleic acid signature specific to a particular threatening biologicalagent or simulant) with a nucleic acid probe capable of selectivelyhybridizing to at least a portion of the target nucleic acid sequence.The chimeras of the present invention are nucleic acid and can bedetected by nucleic acid probes. Nucleic acid hybridization methodsapplicable to this invention are described in Sambrook et al. Thedetection may also occur by polymerase chain reaction (PCR) as describedin Barlett et al.

PCR is typically used in nucleic acid based detection methods. Smallamounts of biological threat agents may be present in a suspect sampleand the corresponding low amount of nucleic acid sequences of thebiological threat agents may have to be amplified to be detected. Inorder to amplify the nucleic acid sequences of a biological threatagent, lysis of the cell, or virus particle, preferably occurs byconventional methods. Then the nucleic acid sequences present in thesample are heated so that it becomes denatured to form single strandednucleic acid sequences. The denatured nucleic acid sequences are cooledand nucleic acid probes are annealed. The probes are specific to thebiological threat agent thought to be in the sample. Taq or equivalentpolymerase binds the 3′ end of each nucleic acid probe annealed tonucleic acid sequences and extends each of these primers in the 5′ to 3′direction along the nucleic acid sequences. PCR typically results in adoubling of the number of copies of nucleic sequences after each roundof DNA synthesis and a geometric increase in number of copies after eachreaction cycle. The chimera in the present invention can be used to testdifferent primers (probes), conditions, specificity, and sensitivity tobe used in the PCR amplification method, or nucleic acid based detectionmethods. The PCR product (amplified nucleic acid sequence) can beobserved afterwards by separation of the DNA by agarose gelelectrophoresis, capillary electrophoresis, real time fluorescence, orother detection methods known to those familiar in the art.

Some biological defense technology must be able to detect very smallamounts of threatening biological agents in a relative large amount ofmaterial; for example, a small number of anthrax spores in a thick layerof dust on top of a computer. Such non-pathogenic material collectedwith a threatening biological agent must be removed before a threateningbiological agent may be detected and identified. Methods for the removalof such non-pathogenic materials may include, but are not limited to,purification by means of ligand-receptor affinity (of whichantibody-antigen affinity or nucleic hybridization are possibleexamples).

Other types of decontamination technologies include but are not limitedto methods and devices that transmit radiant energy (such as ultravioletradiation) to threatening biological agent cells, spores, or virions insuch a way that the absorbance of the radiant energy disruptsthreatening biological agent cells, spores, or virions in the waysmentioned above. Another class of decontamination technology includesmethods or devices that generate aerosols or gaseous emissions ofsubstances that inactivate threatening biological agent cells, spores,or virions in the ways described above. An example of such a technologyis a vaporous hydrogen peroxide (VHP) generator. Hydrogen peroxidevapors, chlorine dioxide, paraformaldehyde vapors, or combinationsthereof, are capable of penetrating the interiors of equipment anddestroying threatening biological agent cells by chemically (oxidativelyor otherwise) modifying small or macromolecules of threateningbiological agent cells, spores, or virions so that they are no longerviable or able to cause disease.

A simulant of the present invention is a chimera containing segments ofnucleic acid sequences, which is safe when in contact with humans and isable to take the place of a biological threat agent, preferably duringthe development, testing, and training of biological defense technology.

EXAMPLES Example 1 Design and Synthesis of a Nucleic Acid Segments forDetecting Biological Threat Agents having DNA Genomes

A single molecule chimera was made of DNA segments, each segmentcorresponding to the nucleic acid sequences of a biological threat agenthaving a DNA genome. The segments were identified using a novelbioinformatics approach. As shown in FIG. 1, this bioinformaticsapproach has multiple steps and uses computational tools to search andselect non-infectious signature sequences corresponding to bacterial andviral threat agents whose genome is DNA, including Bacillus anthracis,Yersinia pestis, Coxielila Bumeti, Brucella sp., Francicella tularensis,Entherohemorragic E. coli, O157:H7, Burkholderia mallei, Burkholderiapseudomallei and Variola virus (smallpox virus).

Once these nucleic acid sequences (or segments of the chimera) wereidentified, each segment was then prepared by PCR amplification.Synthetic chimeras were designed to produce PCR amplicons of differentsizes than the amplified fragments from the original pathogenic genome(to identify any false positives).

Segments of the sizes shown in FIG. 2 were chosen to create the chimerafor detecting Biological Threat Agents having DNA genomes. Added to eachfragment were two restriction sites in the middle of the sequence(EcoRI-GAATTC- and SmaI-CCCGGG-). These enzymes won't cut the amplifiedsegments from the microbial genomes; therefore the enzymes can be usedto digest these segments in case of suspected contamination with thesimulant. When the simulant amplicons were digested with internalrestriction enzymes, two small fragments were obtained. (see right twocolumns in FIG. 2) For example, the Francisella tularensis simulantamplicon was a size of 100 bp and was digested by EcoR1 into twofragments of 37 bp and 63 bp were obtained. The corresponding fragmentin the threat agent Francisella tularensis is 230 by and is not digestedby EcoR1.

Based on the bioinformatics study described in FIG. 1 and the primers(underlined in bold below) designed from segment sequences using theFastPCR software, DNA segments were selected as follows:

Francisella Segment

[SEQ ID NO. 1] GGATCCGACAAGCTTATGGCTTTGC AGCCACTTTTGCAATCGCTGTGTG AGCCCGGGCAGCGAATTCCCATTTAGATTTTTTTGAATATGCTTGTAAA GACCGAGGCTCAGAACTAATCGCAGCT AGAGGACAAGYersinia Segment

[SEQ ID NUMBER: 2] GGATCCTGAAAGCTTGCTGGGGCGA ACCCACCTCATTGGCTATGGCGGC GTCGCCTGTCACGTCCTGTTTGAGTGGGATAAACGCCACGATGAGTTCGATCTCGCCATACTGGAGAAAGGATGGAACCAGCTCATCGCACGCCACGATATGTTGCGTATGGTGGTTGCCCGGGGCCTGAATTCTGAGGATCCTCATTATGTCAATATCGGTACGGTGTTAGACAAGGCCGACTG ACGCCGGAGTA TCACATCCCGCGTGACGATCTGCGCBurkholderia Segment

[SEQ ID NUMBER: 3] GGATCCATGAAGCTTCATTCGTCTT TGCCATTGCCCTGTCATTTGCCGC AGCCCGGGTGCTGAATTCGTCAGCAATGCGAAATTTACATCCCTACGCG AGCCTTTTGTTTTTACCGACCTGAGTCTGTTCAGTCAGTTGT TCTCGCA CCCpXO2 B. Anthracis Segment

[SEQ ID NUMBER: 4] GGATCCCTCAAGCTTTTACACGTTT TGCTGACCAATCTAAGCCTGCGTTCTTCGTAAATGGTTTTGCAGCGAATGATCCCTCATCAACATTACGTATTTGGGAACGTGTGGATGATTTTGGATATAGTAATCTAGCTCCAATTGCCCGGGAGATGAATTCTACATCTGCGCGAATGATATATTGGTTTACTGACGAGGAGCAACCGATTAAGCGCCGTA GCGTTGATCGTACTGAGCAGTTTGCT AGGGATGTTTRickettsia Segment

[SEQ ID NUMBER: 5] GGATCCGGAAAGCTTAGCTGGTATC GCTTATTTTAGAGGTTATAGAGTT CGCCCGGGTAGTGAATTCGTAAACCTTTATTTTTTGATCTTAATATTTCTACTAGAACCCAAAACGTATCCCAAGTTCAA AGAGCTTTACTTTTACCT CAAGAAGT AATACAGTTApXO1 B. Anthracis Segment

[SEQ ID NUMBER: 6] GGATCCTCTAAGCTTGAAAAAGGATTGGAT TTCAAGTTGTACTGGACCG ATTCTCAAAATAAAAAAGAAGTGATTTCTAGTGATAACTTACAATTGCCAGAATTAAAACAAAAATCTTCGAACTCAAGAAAACCCGGGGAAAGAATTCTCATCTCCTGAAAAATGGAGCACGGCTTCTGATCCGTACAGTGATTTC GAAAAGGTTGGACCTACGGTTCCAGACCGTGACA ATGATGGAATCoxiella Segment

[SEQ ID NUMBER: 7] GGATCCACTAAGCTTCGGATTGTTA CCCAACGAAACCTTGCGTGAGGCA TTGAATCGGGAATTAGATGAAGAAGTGGGACTGAGTCCTCACCCGGGTA|CAGAATTCCAATGGCGGTGGGTTGATTATTGGTATCCGGTGGACCA CGTCGTTGAGTTTAAGCGAGACGTTT ATCAGAAAGTVariola Segment

[SEQ ID NUMBER: 8] GGATCCATAAAGCTTCGGAAGAGAT GCAGCACCGTATACACCACCCAAT GGAATCATTAGTATACTCTACACCTTATCCTCAGACACAGATATCTAAAAAAATAGGTGATGATGCAACTCTATCATGTAGTAGAAATAATATACCCGGGACGTGAATTCCAAACAAAATGTGGAATAGGATACGGAGTA TCCGGAC ACACGTCTGTTGGAGACGTCATCTGTTCTBrucella Segment

[SEQ ID NUMBER: 9] GGATCCTAGAAGCTTAATTGTGGGC CGATGGCGTCATCCATGTGCTGG GTGTCGGGCTGGCGCTTGCCGGTGCCATTGCCATGCTGTTCTATTTCCTCCCGGGAATGGAATTCTATGGGCGACCGCGCGGTGCCCCTGCTGCTG TTCGTGTGGAGCGTGGCTTTCGTCGGCATCATGCTCAAACT GTTCATG CCGEscherichia Segment

[SEQ ID NUMBER: 10] GGATCCCTGAAGCTTGCGCGCTAAC GCAGGCCTGAACTCATCGTCGGATGA ATTACAGGCCCAGACGCGTATTGCCGGAATGCGCTCAACGCTGGAGCAATATCACCCGGGGCACGAATTCAAGCGCAATACTGGCCAACGCTCAGTATTCAGGGGGGTAAAACGCGCTACCAG ACCAGCGACCGCTCGTAT TGGGATGA TCAGCTACAASmallpox Segment

[SEQ ID NUMBER: 11] TCATTAGTATACTCTACACCTTATCCTCAGACACAGATATCTAAAAAAATAGGTGATGATGCAACTCTATCATGTAGTAGAAATAATATA

A chimera able to mimic many different types of biological threat agentswas created by DNA synthesis and the joining of the above-identifiedsegments. The whole chimera sequence for DNA genome threat agents is SEQID NUMBER: 12.

[SEQ ID NO: 12] GGATCCGACAAGCTTATGGCTTTGC AGCCACTTTTGCAATCGCTGTGTGAGCCCGGGCAGCGAATTCCCATTTAGATTTTTTTGAATATGCTTGTAAAG ACCGAGGCTCAGAACTAATCGCAGCT ACAGCACAAGGGATCCTGAAAGCTTGCT GGGGCGAACCCACCTCATTGGCTATGGCGGCGT CGCCTGTCACGTCCTGTTTGAGTGGGATAAACGCCACGATGAGTTCGATCTCGCCATACTGGAGAAAGCATGGAACCAGCTCATCGCACGCCACGATATGTTGCGTATGGTGGTTGCCCGGGGCCTGAATTCTGACGATCCTCATTATGTCAATATCGGTACGGTGTTAGA CAACGCCGACTGACGCCGGAGTATCACATCCCGCGTGA CGATCTGCGCGGA TCCATGAAGCTTCATTCGTCTTTGCCATTGCCCTGTCATTTGCCGCAG CCCGGGTGCTGAATTCGTCAGCAATGCGAAATTTACATCCCTACGCGAGCCTTT TGTTTTTACCGACCTGAGTCTGTTCAGTCAGTTGT TCTCGCACCCGGATCC CTCAAGCTTTTACACGTTTTGCTGACCAATCTAAGCCTGCGTT CTTCGTAAATGGTTTTGCAGCGAATGATCCCTCATCAACATTACGTATTTGGGAACGTGTGGATGATTTTGGATATAGTAATCTAGCTCCAATTGCCCGGGAGATGAATTCTACATCTGCGCGAATGATATATTGGTTTACTGACGAGGAGCAACCGATTA AGCGCCGTAGCGTTGATCGTACTGAGCAGTTTGCT AGGGATGTTTGGATCC GGAAAGCTTAGCTGGTATCGCTTATTTTAGAGGTTATAGAGTTCG CCCGGGTAGTGAATTCGTAAACCTTTATTTTTTGATCTTAATATTTCTACTAGAACCCAAAACGTATCCCAAGTTCAA AGAGCTTTACTTTTACCTCAAGAAGT AATACAGTTAGGATCCTCTAAGCTTGAAAAAGGAT TGGATTTCAAGTTGTACTGG ACCGATTCTCAAAATAAAAAAGAAGTGATTTCTAGTGATAACTTACAATTGCCAGAATTAAAACAAAAATCTTCGAACTCAAGAAAACCCGGGGAAAGAATTCTCATCTCCTGAAAAATGGAGCACGGCTTCTGATCCGTACAGTGATTTCGA AAAGGTTGGACCTACGGTTCCAGACCGTGACA ATGATGGAATGGATCCACT AAGCTTCGGATTGTTACCCAACGAAACCTTGCGTGAGGCAT TGAATCGGGAATTAGATGAAGAAGTGGGACTGAGTCCTCACCCGGGTACAGAATTCCAATGGCGGTGGGTTGATTATTGGTATCCGGTGGACCA CGTCGTTGAGTTTAAGCG AGACGTTTATCAGAAAGTGGATCCATAAAGCTTCGGAAGAGAT GCAGCACC GTATACACCACCCAATGGAATCATTAGTATACTCTACACCTTATCCTCAGACACAGATATCTAAAAAAATAGGTGATGATGCAACTCTATCATGTAGTAGAAATAATATACCCGGGACGTGAATTCCAAACAAAATGTGGAATAGGATACGGA GTATCCGGACACACGTCTGTTGGAGACGT CATCTGTTCTGGATCCTAGAAG CTTAATTGTGGGCCGATGGCGTCATCCATGTGCTGGGTG TCGGGCTGGCGCTTGCCGGTGCCATTGCCATGCTGTTCTATTTCCTCCCGGGAATCGAATTCTATGGGCGACCGCGCGCTGCCCCTGCTGCTGTTCGTGTGGAGCGTG GCTTTCGTCGGCATCATGCTCAAACT GTTCATGCCGGGATCCCTGAAGCTTGCGCGC TAACGCAGGCCTGAACTCATCGTCGGATGA ATTACAGGCCCAGACGCGTATTGCCGGAATGCGCTCAACGCTGGAGCAATATCACCCGGGGCACGAATTCAAGCGCAATACTGGCCAACGCTCAGTATTCAGGGGGGTAAAACGCGCTACCAGACCAGCGACCGCTCGTATTGGGATGA TCAGCTACAAAAGCTTAGAGGATCC

A plasmid map comprising the whole chimera is shown in FIG. 4.

Example 2 Design and Synthesis of a Nucleic Acid Segments for DetectingBiological Threat Agents having RNA Genomes

The strategy used to identify nucleic acid segments unique to BiologicalThreat Agents was different than that used in Example 1. The reason isthat there is a higher probability of finding a unique DNA in largerbacterial genomes (Example 1) than in smaller viral genomes due to thesignificant disparity in genomic size between bacteria and viruses.Smaller viral genomes (Example 2) have been sequenced completely, unlikebacterial genomes requiring the need of large sequencing efforts. Toobtain segments, or conserved regions of nucleic acid, among allisolates of one viral species, the genome sequences from all availableisolates were aligned using ClustalW software (Thompson, J. D. et al1997). The selection of possible primer sequences was performed manuallylooking at the alignments. This analytical approach was used todetermine target nuclei acid sequence representing several RNA viruswhose genome is RNA, including but not limited to, nucleic acids in VEEV(Venezuelan Equine Encephalitis Virus), Influenza virus, Rift ValleyFever Virus, Machupo virus, Lassa virus, Yellow Fever virus, Ebola Zairevirus, Eastern Equine Encephalitis Virus, Junin virus, Marburg virus,Dengue virus, Crimean Congo virus.

Primer sequences were then selected manually by looking at the sequencealignments. Then Fast PCR was used as described in Example 1.

The following DNA Sequences were selected, based on the manual selectiondescribed above, and primers (underlined in sequences below) weredesigned from segment sequences using the FastPCR software for purposesof designing and chemically synthesizing the whole chimera as follows:

Restriction Sites:

GAATTCTACCCCGGG EcoRI/SmaI (intrafragments sites)

AAGCTTCGCGGATCC HindIII/BamHI (interfragments sites)

Ebola Segment

[SEQ ID NUMBER: 13] AAGCTTCGCGGATCCCG GCAATTGCACTCGGAGTCGCCACAGCACACGGGA GTACCCTCGCAGGAGTAAATGTTGGAGAACAGTATCAACAACTCAGAGAGGCTGCCACTGAGGCTGAGAAGCAAGAATTCTACCCCGGG TGCTGCGTCACTG CCCAAAACAAGTG GAEEEV Segment

[SEQ ID NUMBER: 14] AAGCTTCGCGGATCCTTT ACTTGTCTGCGGCGCCTTGGGCGCCGTAGTCGA ACGCCCAGGTTATGCACCCGTTCACCTACAGATACAGCTGGTTAATACCAGGATAATTCCATCAAGAATTCTACCCCGGGACAGGTGTTTACCCATTCATGT GGGGAGGAGCCTACTGCTTCTGCG ACJunin Segment

[SEQ ID NUMBER: 15] AAGCTTCGCGGATCCGC ACCTCTGATCCAGACATGCAGTCGACCCTTAACT TTGACATCAAATCCACATGATGGATTTGATTTGCATATGCCATCAAGAAATATCTTAGACCTTGTAAAAATGTCTGGTTCCGAATTCTACCCCGGGCCCATT GATGGATAGATAGATAGAATAGCACCTTGACTTCTCACCTGTTT TTMarburg Segment

[SEQ ID NUMBER: 16] AAGCTTCGCGGATCCAT GAAGTTGCTAGTTTCAAGCAGGCGTTGAGCAACC TAGCCCGACATGGAGAATACGCACCGTTCGCACGGGTTCTGAATTTATCAGGGATTAACAACCTCGAACATGGACTCTATCGAATTCTACCCCGGGTTCAGAAAACTGAAATCACACACAGTCAGACACTA GCCGTCCTCAGCCAGAAACGAG AAA AADengue Segment

[SEQ ID NUMBER: 17] AAGCTTCGCGGATCCTT TCAATATGCTGAAACGCGAGAGAAACCGCGTGTC AACTGTTTCACAGTTGGCGAAGAGATTCTCAAAAGGATTGCTTTCAGGCCAAGGACCCATGAAATTGGTGATGGCTTTTATAGCGAATTCTACCCCGGGTTATGTGAGGACACAATGACCTACAAATGCCCCCGGATCACTGAGACG GAACCTGAAGACATTGACTGTTGGTGCAA TGVEEV Segment

[SEQ ID NUMBER: 18] AAGCTTCGCGGATCCTA GTTAGTTGCGACGGGTACGTCGTTAAAAGAATAG CTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGTTAAAAGAATAGCTATCAGGAATTCTACCCCGGGGGCTATGCTGCTACGATGCACCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCAC CGCGAGGGATTCTTGTGCTGC AACrimean Congo Segment

[SEQ ID NUMBER: 19] AAGCTTCGCGGATCCAA TTGATGATGAGCATGTCAGGCATTGATTGTATAA AATATCCCACAGGGCAGCTTATCACCCATGGAAGAGTGAGTGCAAAACATAACGATGGGAACCTGAAAGATAGAAGCGAGAATTCTACCCCGGGAACCTGTGCCCTTTCAGGTTGACTGTATATTGTTCAAAGAAGTGGCAGCTGAATGCATG AAGAGGTACATTGGCACACCTTATGAGGGAATT GTInfluenza Segment

[SEQ ID NUMBER: 20] AAGCTTCGCGGATCCAA ACCATTTGAATGGATGTCAATCCGACTCTACTGT TCCTAAAGGTTCCAGCGCAAAATGCCATAAGCACCACATTCCCTTATACTGGAGATCCTCCATACAGCCATGGAACAGTCTACTGTTGAATTCTACCCCGGGTGGAACAGTCTACTGTTCCTAAAGGTTCCAGCGCAAAATGCCATAAGCACCACATTCCCTTATACTGGAGATCCTCCATACAGCCATGGAACAG GAACAGGA TACACCATGGACACAGTCAARVFV Segment

[SEQ ID NUMBER: 21] AAGCTTCGCGGATCCTT ATGAGTGCACTGCTCAGTACGCCAATGCCTATTG TTCACATGCTAATGGGTCAGGGATTGTGCAGATACAAGTATCAGGGGTCTGGAAGAAGCCTTTATGTGTAGGGTATGAGAGAGTGGTTGTGAAGAGAGGAATTCTACCCCGGGACATGCTAATGGGTCAGGGATTGTGCAGATACAAGTATCAGGGGTCTGGAAGRAGCCTTTATGTGTAGGGTATGAGAGAGTGGTTGTGAAG AGAGAACTCTCTGCCAAGCCCATCCAGAGAGTTGAGCCTTGC ACMachupo Segment

[SEQ ID NUMBER: 22] AAGCTTCGCGGATCCTT CATTCATCATGTCTAAAGCAATGCAGACATCCAG AAATTTTAGCCTCCCGCTATCCATTGTTCTGCTGACCTGAAGATCATTCATAAATGGAGTCAAGTGTTCGTCAAAAAGAACTGGATAATTTCTCCTTATAGATTGAATTCTACCCCGGGTCTGCTGACCTGAAGATCATTCATAAATGGAGTCAAGTGTTCGTCAAAAAGAACTGGATAATTTCTCCTTATAGATTGCAGAACATGGTTCATTCCCAGTTGGTCTTCAATTTG TCTCACCACTTTAGGCTTCACA GCC CALassa Segment

[SEQ ID NUMBER: 23] AAGCTTCGCGGATCCTT ATCCTGGGTGACCACTTCATTTTGGTTGATGCTA AGTCGCTCATAAATGGCAGTATGTGTTTTTCAAATACAGATGGGAATTCTACCCCGGG AAGACCCATGCACCCAGTTCTATTGC AGYellow Fever Segment

[SEQ ID NUMBER: 24] AAGCTTCGCGGATCCTG CTAAGCTGTGAGGCAGTGCAGGCTGGGACAGCCG ACCTCCAGGTTGCGAAAAACCTGGTTTCTGGGACCTCCCACCCCAGAGTAAAAGAATTCTACCCCGGG CAGTTTGCTCAAGAATAAGCAGACCT TTActin Segment (450 pb)

[SEQ ID NUMBER: 25] AACCTTCGCGGATCCGCGTCCGCCC CGCGAGCACAGAGCCTCGCCTTTGCCGATCCGCCGCCCGTCCACACCCGCCGCCAGCTCACCATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCGAATTCTACCCCGGGTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAACTGGGACGACATGGAGAAAATCTGGCACCACACCTTCTACAATGAGCTGCGTGTGGCTCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCGC GAGAAGATG ACCCAGATCATGTTTGAGACCTTCAA

These segments were then joined together to form a chimera to mimic manydifferent types of biological threat agents whose genome is RNA. DNAsynthesis was used to create the whole chimera based on the joining ofsegments. The entire chimera sequence for threat agents having RNAgenomes is SEQ ID NO: 26.

[SEQ ID NUMBER: 26] AAGCTTCGCGGATCCTT ATCCTGGGTGACCACTTCATTTTGGTTGATGCTA AGTCGCTCATAAATGGCAGTATGTGTTTTTCAAATACAGATGGGAATTCTACCCCGGG AAGACCCATGCACCCAGTTCTATTGC AGAAGCTTCGCGGATC CGCGTCCGCCCCGCGAGCACAGAGCC TCGCCTTTGCCGATCCGCCGCCCGTCCACACCCGCCGCCAGCTCACCATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCTTCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGATGGTGGGCATGGGTCAGAAGGATTCCGAATTCTACCCCGGGTATGTGGGCGACGAGGCCCAGAGCAAGAGAGGCATCCTCACCCTGAAGTACCCCATCGAGCACGGCATCGTCACCAACTGGGACGACATGGAGAAAATCTGGCACCACACCTTCTACAATGAGCTGCGTGTGGCTCCCGAGGAGCACCCCGTGCTGCTGACCGAGGCCCCCCTGAACCCCAAGGCCAACCG CGAGAAGATGACCCAGATCATGTTTGAGACCTTCAAAAGCTTCGCGGATCCTGC TAAGCTGTGAGGCAGTGCAGGCT GGGACAGCCGACCTCCAGGTTGCGAAAAACCTGGTTTCTGGGACCTCCCACCCCAGAGTAAAAGAATTCTACCCCGGG CAGTTTGCTCAAGAATAAGCAGAC CTTTAAGCTTCGCGGATCCTT CATTCATCATGTCTAAAGCAATGC AGACATCCAGAAATTTTAGCCTCCCGCTATCCATTGTTCTGCTGACCTGAAGATCATTCATAAATGGAGTCAAGTGTTCGTCAAAAAGAACTGGATAATTTCTCCTTATAGATTGAATTCTACCCCGGGTCTGCTGACCTGAAGATCATTCATAAATGGAGTCAAGTGTTCGTCAAAAAGAACTGGATAATTTCTCCTTATAGATTGCAGAACATGGTTCATTCCCAGTTGGTCTTCAATTTG TCTCACCACTTTAGGCTT CACAGCCCAAAGCTTCGCGGATCCCG GCAATTGCACTCGGAGTCGCCACAG CACACGGGAGTACCCTCGCAGGAGTAAATGTTGGAGAACAGTATCAACAACTCAGAGAGGCTGCCACTGAGGCTGAGAAGCAAGAATTCTACCCCGGG TGCTGCGTCACTGCCCAAAACAAGTG GAAAGCTTCGCGGATCCTT ATGAGTGCAC TGCTCAGTACGCCAATGCCTATTGTTCACATGCTAATGGGTCAGGGATTGTGCAGATACAAGTATCAGGGGTCTGGAAGAAGCCTTTATGTGTAGGGTATGAGAGAGTGGTTGTGAAGAGAGGAATTCTACCCCGGGACATGCTAATGGGTCAGGGATTGTGCAGATACAAGTATCAGGGGTCTGGAAGAAGCCTTTATGTGTAGGGTATGAGAGAGTGGTTGTGAAGAGAGAACTCTCTGCCA AGCCCATCCAG AGAGTTGAGCCTTGCACAAGCTTCGCGGATCCTT TACTTGTCTGCGGCGCC TTGGGCGCCGTAGTCGAACGCCCAGGTTATGCACCCGTTCACCTACAGATACAGCTGGTTAATACCAGGATAATTCCATCAAGAATTCTACCCCGGGACAGG TGTTTACCCATTCATGTGGGGAGGAGCCTACTGCTTCTGCG ACAAGCTTCG CGGATCCAA ACCATTTGAATGGATGTCAATCCGACTCTACTGTTCCTAAAG GTTCCAGCGCAAAATGCCATAAGCACCACATTCCCTTATACTGGAGATCCTCCATACAGCCATGGAACAGTCTACTGTTGAATTCTACCCCGGGTGGAACAGTCTACTGTTCCTAAAGGTTCCAGCGCAAAATGCCATAAGCACCACATTCCCTTATACTGGAGATCCTCCATACAGCCATGGAACAG GAACAGGATACACCAT GGACACAGTCAAAAGCTTCGCGGATCCGC ACCTCTGATCCAGACATGCAGT CGACCCTTAACTTTGACATCAAATCCACATGATGGATTTGATTTGCATATGCCATCAAGAAATATCTTAGACCTTGTAAAAATGTCTGGTTCCGAATTCTACCCCGGGCCCATTGATGGATAGATAGATAGAAT AGCACCTTGACTTCTCACC TGTTTTTAAGCTTCGCGGATCCTAGTTA GTTGCGACGGGTACGT CGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGTTAAAAGAATAGCTATCAGGAATTCTACCCCGGGGGCTATGCTGCTACGATGCACCGTTAAAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCAC CGCGAGGGATTCTTGTGCTG CAAAAGCTTCGCGGATCCAT GAAGTTGCTAGTTTCAAGCAGGCGT TGAGCAACCTAGCCCGACATGGAGAATACGCACCGTTCGCACGGGTTCTGAATTTATCAGGGATTAACAACCTCGAACATGGACTCTATCGAATTCTACCCCGGGTTCAGAAAACTGAAATCACACACAGTCAGACACTA GCCGTCCTCAGCCAGAAAC GAGAAAAAAAGCTTCGCGGATCCAA TTGATGATGAGCATGTCAGGCAT TGATTGTATAAAATATCCCACAGGGCAGCTTATCACCCATGGAAGAGTGAGTGCAAAACATAACGATGGGAACCTGAAAGATAGAAGCGAGAATTCTACCCCGGGAACCTGTGCCCTTTCAGGTTGACTGTATATTGTTCAAAGAAGTGGCAGCTG AATGCATGAAGAGGTACATTGGCACACCTTATGAGGGAATT GTAAGCTTCG CGGATCCTTTCAATATGCTGAAACGCGAGAGAAACCG CGTGTCAACTGTTTCACAGTTGGCGAAGAGATTCTCAAAAGGATTGCTTTCAGGCCAAGGACCCATGAAATTGGTGATGGCTTTTATAGCGAATTCTACCCCGGGTTATGTGAGGACACAATGACCTACAAATGCCCCCGGATCACTGAGACG GAACCTGAAGACAT TGACTGTTGGTGCAATGAAGCTTCGCGGATCCSize: 3143 bp

Once these nucleic acid sequences (or segments of the chimera) wereidentified, each segment was then prepared by PCR amplification.Synthetic chimeras were designed to produce PCR amplicons of differentsizes (as indicated in FIG. 3) than the amplified fragments from theoriginal pathogenic genome (to prevent that any contamination withstimulant could create false positives).

The chimera containing sequences corresponding to Biological ThreatAgents having RNA genomes was inserted in the plasmid vector pBluscriptSKII. A plasmid drawing comprising the whole chimera is described inFIG. 5, that shows the location in the plasmid vector of segmentsspecific to each biothreat agent (separated by a Barn H1 restrictionsite), as well as the positions of restriction enzymes (SacI and XhoI)at the extremes of the insert.

The correct design and construction of the chimerical simulants (one forDNA agents and the other for RNA agents) was experimentally confirmed byreleasing the inserts from the plasmid vector by digestion with one ofthe intersegment restriction enzymes (BamH1), performing multiplex PCR(using as primers the oligonucleotides underlined in sequences 1-26),and subsequent electrophoretic analysis shown in FIG. 6. The twovertical columns pointed by arrows in the gel in FIG. 6 correspond tonucleic acid fragments of the expected size (as indicated in FIG. 3) foragents whose genome is RNA (bands in column pointed by short downwardarrow), and nucleic acids of the expected size (as indicated in FIG. 2)for agents whose genome is DNA (bands in column pointed by long downwardarrow). The names of the agents are aligned to the correspondingfragments and their sizes are indicated (in base pairs, bp) at each sideof the image representing the gel electrophoresis analysis.

REFERENCES

-   Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular    Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory    Press, Cold Spring Harbor, N.Y.-   Bartlett J. M. S., Stirling D., eds. 2003. PCR Protocols, 2^(nd) ed.    (Volume 226 in the series Methods in Molecular Biology.) Humana    Press, Totowa, N.J.-   Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., and    Higgins D. G. The CLUSTAL_X windows interface: flexible strategies    for multiple sequence alignment by quality analysis tools. Nucleic    Acids Res. 1997 Dec. 15; 25(24): 4876-82.

The foregoing description of embodiments of the present inventionprovides an exemplary illustration and description, but is not intendedto be exhaustive or to limit the invention to the precise formdisclosed. Modifications and variations are possible in light of theabove teachings or may be acquired from practice of the invention.

1. A chimera comprising a plurality of segments, wherein each segmentuniquely corresponds to a portion of the genome of a biological threatagent whose genome is RNA, and wherein said plurality of segmentscomprises a nucleic acid sequence comprising SEQ ID NO:
 26. 2. A vectorcomprising the chimera of claim
 1. 3. The vector of claim 2, wherein thevector is selected from the group consisting of a plasmid, a virus, acosmid, or a yeast artificial chromosome.
 4. The vector of claim 3,wherein the vector is a plasmid.